Abstract Chronic pain conditions cause an immense burden on society due to their astonishingly high prevalence and lack of effective treatments. This application addresses how indirect modulation of the excitability of neurons in pain conditions can be achieved by altering the expression and function of the Nav1.7 sodium channel. The scientific premise is that because direct blockade of Nav1.7 channels has been unsuccessful, targeting regulators of Nav1.7 may offer therapeutic advantages allowing for a graded analgesic response. Dr. Rajesh Khanna, Principal Investigator on this project, first discovered that expression of Nav1.7 at the surface is regulated by a protein, axonal collapsin response mediator protein 2 (CRMP2), and that a mutant of CRMP2 lacking the small ubiquitin-like modifier (SUMO) post-translational modification (deSUMOylation) reduces Nav1.7 surface expression and currents. Importantly, the related Nav1.1, Nav1.3, Nav1.5, Nav1.6, Nav1.8, and Nav1.9 channels are unaffected. In preliminary studies, we demonstrate that loss of CRMP2 SUMOylation increases binding to endocytic proteins, potentially accounting for removal of Nav1.7 from the surface. The fraction of SUMOylated CRMP2 increases significantly following peripheral nerve injury. Excitingly, in vivo transfection of a CRMP2-K374A SUMO-null plasmid or a peptide mimicking the CRMP2 SUMOylation motif, into the spinal cord reversed mechanical allodynia in a model of neuropathic pain. Together, these findings strongly support the hypothesis that loss of CRMP2 SUMOylation reduces Nav1.7 localization at the plasma membrane, thereby decreasing nociceptive neuron excitability and thresholds to thermal and mechanical stimuli in acute and chronic pain. We will test this hypothesis in three specific aims. In Aim 1, we will test the general role of CRMP2 SUMOylation on Nav1.7 currents and neuronal excitability using a recently created new transgenic K374A Crmp2 knock-in mouse model where the SUMOylation site (K374) of CRMP2 has been replaced with an alanine mutation; this mouse was made by Dr. Thomas Doetschman, a co-Investigator on this project and Director of the Genetically Engineered Mouse Models Core facility at the University of Arizona. The mechanism by which Nav1.7 surface trafficking and internalization occur is unknown and will be examined in Aim 2 of this proposal. Aim 3 will evaluate the contribution of the CRMP2 SUMOylation state to acute pain thresholds as well as after experimentally induced pain thresholds using models in which Nav1.7 levels are increased; these studies will be performed in collaboration with Dr. Todd Vanderah, a co-Investigator on this project with deep expertise in preclinical pain modeling. Finally, in these mice, we will also measure CRMP2- dependent off-target effects on memory, locomotion/sedation, as well as behaviors linked to Nav1.7, including smell. The proposed study will considerably improve our understanding of how intracellular trafficking proteins can be modified in diseases/injuries, lay a solid foundation for unraveling mechanisms of the modification and trafficking of Nav1.7 in chronic pain, and offer novel and selective therapeutic targets for pain research.